Antibodies, particularly humanized antibodies, have become very useful for diagnostic and therapeutic purposes. Humanized antibodies are antibodies in which CDRs or hypervariable regions (HVRs) from a non-human antibody are combined with human framework regions to form an antigen binding molecule. This exchange is sometimes known as a “CDR swap”. There are different ways of selecting human framework sequences for humanized antibodies. One method involves selecting a human variable domain sequence that has a very similar framework sequence to that of the non-human antibody that is the source of the CDRs. Another method involves using a human variable domain consensus sequence as the source of the human framework regions. Often, a straight CDR swap does not result in high affinity antigen binding molecules so that additional changes or modifications are required to improve binding affinity of the humanized antibody. The necessity of making additional modifications can make humanization of antibodies a very time consuming process. In addition, humanization may not result in an antibody that can be produced in high yield in cell culture.
Some of the uses of antibodies require large quantities of full-length completely assembled antibodies. Many techniques are now available for producing antibodies recombinantly using a variety of host cell systems including E. coli, yeast, plant cells, insect cells, and mammalian cells. Eukaryotic and prokaryotic systems have been used in large-scale production of antibodies. In particular, E. coli provides a useful organism for the expression of antibodies, including engineered antibodies, such as humanized antibodies. There are several advantages to E. coli expression systems, including a well-studied and convenient gene technology which permits constructs to be made easily and directly expressed, and the relatively convenient and economical large-scale production of product made possible by the fast growth of E. coli and its comparatively simple fermentation.
Full-length antibodies comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to one of the heavy chains by a disulfide bond. Each chain has an N-terminal variable domain (VH or VL) and one or more constant domains at the C-terminus; the constant domain of the light chain is aligned with and disulfide bonded to the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Each of the variable domains of the heavy and light chain includes framework regions (FRs) and hypervariable regions (HVRs) and an intrachain disulfide bond. (See e.g. Chothia et al., J. Mol. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:45924596 (1985); Padlar et al., Mol. Immunol., 23(9): 951-960 (1986); and S. Miller, J. Mol. Biol., 216:965-973 (1990). Antibody fragments are also often produced and include combinations of heavy and light chain variable domains so as to form an antigen binding site. Antibody fragments include, for example, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, and Fd′ fragments.
Generally, antibody production in prokaryotes involves synthesis of the light and heavy chains in the cytoplasm followed by secretion into the periplasm where processing of the chains takes place. Alternatively, the heavy and light chains can be directed to accumulate in the cytoplasm where they typically form inclusion bodies. Folding of the light and heavy chains occurs in conjunction with assembly of the folded light and heavy chains to form an antibody molecule. Multiple covalent and non-covalent interactions occur between and within the heavy and light chains during these folding and assembly processes. Antibody yield can be greatly affected by the efficiency and fidelity of these processes. Following synthesis of the heavy and light chains, protein aggregation or proteolysis can occur thereby reducing the yield of the antibody.
Production and stability of antibody fragments have been studied more extensively than that of full length antibodies. Often the stability and/or production yields of scFv or Fab fragments of natural antibodies produced in host cells have been found to be insufficient. Honneger et al., J. Mol. Biol., 309:687-699 (2001). Stability of the antibody or antibody fragment when incubated under physiological conditions is important for therapeutic efficacy in vivo. Production yields and folding efficiency are important to increase the yield of antibodies or antibody fragments for therapeutic use. The stability of scFv fragments is not always correlated with expression yield in the bacterial periplasm. Worn et al., J. Mol. Biol., 305:989-1010 (2001). Some stable scFv fragments show only poor expression yields in bacterial periplasm and some mutations can affect in vivo folding efficiency but not stability. Worn et al., supra. The many factors that affect the periplasmic expression yield and/or stability of scFv are not yet fully understood.
Some structural features thought to be involved in stability and/or in vivo folding of antibody fragments have been previously described. For example, the FR1 of antibody fragments has been found to influence in vivo folding of antibody fragments in bacteria. de Haard et al., Prot. Eng., 11: 1267-1276 (1998). In particular, the data of de Haard et al. suggested that mutations at residue 6 in the heavy chain interfered with the correct folding of a scFv. de Haard et al. supra. Jung et al. have described four different conformations of the FR1 based on the amino acids found at positions H6, H7 and H10 (H9). Jung et al. J. Mol. Biol., 309:701 (2001). Mutations at these residues, especially at residue 6, that disrupt the FR1 conformation can have adverse effects on folding yields and stability of scFv. Jung et al. supra. Residue 6 in the heavy chain is also thought to contribute to the stability of Fab, Fv, and ScFv fragments lacking disulfide bonds. Langdyk et al., J. Mol. Biol., 283:95 (1998). Disulfide bonds also contribute to the stability of antibody domains. When disulfide bonds are removed, the H6 residue helped to stabilize the scFv but not when the disulfide bond was restored. A number of other point mutations at residues have been described as stabilizing or destabilizing in specific scFv fragments. Worn et al., supra. However, the effect of a mutation at a specific residue in an antibody or antibody framework may be unpredictable and may or may not affect the in vivo folding efficiency. A mutation at a residue in one antibody or antibody fragment that is beneficial for folding efficiency and yield may not be beneficial in another.
Methods for producing high affinity humanized antibodies can be time consuming and result in an antibody that is not optimal for production in cell culture. Multiple factors affect the yield and/or stability of antibodies and/or antibody fragments when produced in cell culture. Many of these factors are not yet well understood and may be unpredictable. Thus, there remains a need for improving the process of producing humanized antibodies and for improving the yield of antibodies or antibody fragments in cell culture, especially bacterial cell culture.